Bleomycin and processes for the preparation thereof

ABSTRACT

An antibiotic complex designated bleomycin was produced by fermentation of Streptomyces verticillus A.T.C.C. 15003 and found to inhibit the growth of Gram-negative and Gram-positive and acid-fast bacteria as well as certain plant pathogens and to inhibit the growth of HeLa cells in tissue culture and to inhibit the growth of certain experimental tumors in mice.

United States Patent Umezawa et al.

[151 3,681,491 [4 1 Aug. 1, 1972 BLEOMYCIN AND PROCESSES FOR THE PREPARATION THEREOF Inventors: Hamao Umezawa, 23 Kita-4-chome Toyotama, Nerima-ku, Tokyo; Kenji Maeda, 258 Gotanda-lchome, Shinagawa-ku, Tokyo; Yoshiro Okami, 18-3 Denenchofu-6; Tomio Takeuchi, 273 Imaizumicho, both of Ota-ku, Tokyo, all of Japan Filed: Dec. 3, 1965 Appl. No.: 511,448

Related U.S. Application Data Continuation-in-part of Ser. No. 345,233, Feb. 17, 1964, abandoned.

Foreign Application Priority Data March 5, 1963 Japan ..38/10177 U.S. Cl ..424/115, 195/80 Int. Cl. ..A6lk 21/00 [58] Field of Search ..167/65 AB; 195/80; 424/1 15 [56] References Cited OTHER PUBLICATIONS The Pfizer Handbook of Microbioc Metabolities, l96l,page60l.

Primary Examiner-Jerome D. Goldberg Attorney-Curtis W. Carlson, Richard H. Brink, Robert B. Simonton and Herbert W. Taylor, Jr.

[5 7] ABSTRACT An antibiotic complex designated bleomycin was produced by fermentation of Streptomyces verticillus A.T.C.C. 15003 and found to inhibit the growth of Gram-negative and Gram-positive and acid-fast bacteria as well as certain plant pathogens and to inhibit the growth of HeLa cells in tissue culture and to inhibit the growth of certain experimental tumors in mlce.

7 Claims, N0 Drawings ULTRAVIOLET ABSORPTION SPECTRA OF BLEOMYCIN A AND BLEOMYCIN B WAVE L ENGTH m PATYEMQ Y @972 3 6 81, 491 sum 10? 4 ULTRAVIOLET ABSORPTEON SPECTRA 0F BLEQMYCBN A AND BLEOMYQQN B 2% Sam WAVE LENGTH m):

FIG.I

HAMAO UMEZAWA KENJI MAEDA YOSHIRO OKAMI TOMIO TAKEUCHI INVENTOR.

BY cuR'rns w. CARLSON RICHARD H. BRINK AND HERBERT wmnowm ATTORNEYS PATETEmus 1 m 3,881,

SHIN 3 OF 4 ULTRAVIOLET ABSORPTION SPECTRA OF BLEOMYCIN Cu-Af2 BLEOMYCIN Cu-ATS AND BLEOMYCiN Cu-B Z 0 /Q E I 2 4O 2 8O 2 BO 300 320 340 360 WAVE LENGTH my FIG. 3

HAIVIAO UMEZAWA K ENJI MAE DA BY CURTIS W. CARLSON RICHARD H. BRINK HERBERT W. TAYLOR JRv ATTORNEYS PATENTED 1 9 A w W A M Z A EDKEE M O I U AW OTH O R A WWN mmww v 91 HKYT Onw 00h 00m 00m Q09 00: 00m. O02 O02 OOQ O0: O09 O09 OOON 00mm OOOm 00mm NOISSIWSNVUL 1 NHOBB d BY CURTIS W. CARLSON RICHARD H.BRINK HERBERT W.TAYLOR JR.

ATTORNEYS BLEOMYCIN AND PROCESSES FOR THE PREPARATION F This application is a continuation-in-part of our prior, copending application Ser. No. 345,233, filed Feb. 17, 1964 and now abandoned.

This invention relates to a new antibiotic, bleomycin, and its salts in the pure and the crude state and processes for their production and their isolation, especially their production by the cultivation of microorganisms followed by extraction and isolation. The present invention provides bleomycin as its base or acid addition salt either in purified or crude state and either in the solution or the solid state. Bleomycin inhibits the growth of Grammegative and Gram-positive bacteria including acid-fast bacteria. It exhibits a therapeutic effect against experimental infection of mice with pathogenic bacteria and against experimental animal tumors. On the basis of fundamental studies, it promises to be useful for therapy against bacterial infection in man or animals. In addition, it is inhibitory against the growth of plant-pathogenic organisms and it is effective in prevention or suppression of diseases of rice, pea, wheat or other plants.

There is now provided, according to the present invention, an antibiotic substance effective in inhibiting the growth of Gram-positive bacteria, Gram-negative bacteria, plant pathogens and animal tumors, selected from the group consisting of bleomycin A and bleomycin B, and the acid addition salts thereof, each of said bleomycins being a substance which is soluble in water and methanol and substantially insoluble in ethanol, butanol, acetone, ethylacetate, ether, chloroform and benzene, which forms salts with acids, which exhibits maxima at 244 mp. and 295 my. in the ultraviolet absorption, which gives positive Pauly, Ehrlich and Dragendorf tests and negative Fehling, Tollens, Anthrone and ferrich chloride tests, while bleomycin A1, A2, A3, B1, B2, B3, B4 and B5 give a negative ninhydrin test and A4, A5 and A6 give a positive ninhydrin test, while all bleomycins A, that is Al, A2, A3, A4, A5 and A6, give a negative Sakaguchi reaction and all bleomycins B, that is B1, B2, B3, B4, B5, give a positive Sakaguchi reaction and which contain carbon, hydrogen, nitrogen, sulfur and oxygen with or without copper.

FIG. 1 shows the ultraviolet absorption spectra of bleomycin A and bleomycin B.

FIG. 2 shows the infrared absorption spectra of bleomycin A and B.

As later described, further purification indicated that bleomycin A could be separated into six components designated Al, A2, A3, A4, A5, A6, among which usually A2 and A5 were the main components and that bleomycin B could be separated into B1, B2, B3, B4, B5, among which B2 and B4 were the main components. FIGS. 3 and 4 show the ultraviolet and infrared absorption spectra, respectively, of bleomycin Cu-At2, bleomycin Cu-AtS and bleomycin Cu-Bt2.

There is further provided according to the present invention, a process for the production of bleomycin which comprises cultivating a strain of Streptomyces verzicillus in a medium until a sufiicient amount of bleomycin is accumulated. The bleomycin-producing organism was isolated by the present inventors from a soil sample collected at the coal-mine in Kaho-Gun, Fukuoka-prefecture, Japan. The isolate producing bleomycin was numbered as B-Z2 by the inventors and it was deposited in Division of Mycology, Department of Antibiotics, National Institute of Health, Japan, under the strain number NIHJ 424. This strain was deposited in the American Type Culture Collection, Washington, DC, under the ATCC accession number 15003.

This strain (B80-Z2) possessed the following characteristics: 1. Microscopic characteristics:

Aerial mycelium is developed from fine branched vegetative mycelium. Aerial mycelium forms whorls. In general, the photography of spores on whorls by an electron microscope is difficult. But, in the case of this strain, the spore surface seemed to be smooth.

2. Cultural characteristics on various media:

1. On Czapek-Dox agar plate, incubated at 27 C.:

Pale yellowish-brown growth with good formation of white aerial mycelium. Scant brownish-yellow soluble pigment.

2. On Krainskys glucose asparagine agar plate, incubated at 27 C.:

Growth is pale brownish with yellowish tinge. Good formation of aerial mycelium colored with pale dull greenish gray. No soluble pigment.

3. On starch agar plate, incubated at 27 C.:

Growth is yellowish. Aerial mycelium is white to grayish-white. Slight brownish soluble pigment. None or weak hydrolysis of starch around the growth.

4. On calcium malate agar plate, incubated at 27 C.:

Colorless growth with scant white aerial mycelium.

No soluble pigment.

5. In peptone solution containing 0.2% NaNO incubated at 37 C.:

Pale brownish-yellow growth is formed on the liquid surface as the ring along with the test tube. Also a small amount of mycelium mass is observed at the bottom. White to pale brownish or yellowish-gray aerial mycelium on the surface growth. No soluble pigment. Nitrite from nitrate is detected by the starch-iodine reaction.

6. On bouillon agar slant, incubated at 37 C.:

Colorlessto cream-colored growth with occasional formation of white aerial mycelium. No soluble pigment.

7. On Loefflers coagulated serum slant, incubated at 37 C.: Colorless to cream-colored growth with scant white aerial mycelium. No soluble pigment and no liquefaction of coagulated serum.

8. On blood agar plate, incubated at 37 C.:

Reddish-brown growth with white aerial mycelium.

No soluble pigment and no hemolysis.

9. On gelatin stab, incubated at l820 C.:

Colorless to pale brownish growth. White aerial mycelium. No soluble pigment and no liquefaction of gelatin.

10. On egg medium slant, incubated at 37 C.:

Cream-colored growth with abundant, snowy-white aerial mycelium. No soluble pigment.

l 1. On skimmed milk, incubated at 37 C.:

Cream-colored growth white aerial mycelium on the liquid surface along with test tube. No soluble pigment. Weak coagulation and no or weak peptonization.

12 On potato plug, incubated at 27 C.:

Yellowish-brown growth with powdery white to gray aerial mycelium with brownish tinge. No soluble pigment.

13. On carrot plug, incubated at 27 C.:

Cream-colored growth with brownish tinge. White snowy aerial mycelium. No soluble pigment.

l4. Carbohydrate utilization for growth on Czapeks basal agar medium:

Dextrin, glycerol, starch, glucose and maltose are utilized for good growth. Fructose, inositol and mannose give varied results. No or scant growth was given by arabinose, galactose, inulin, lactose, mannitol, raffinose, rharnnose, salicin, sorbitol, sorbose, sucrose, xylose, sodium acetate, sodium citrate and sodium succinate.

Summarizing the above, the strain B80-Z2 belongs to the genus Streptomyces, and is characterized by formation of whorl, pale brownish-yellow growth, white to grayish aerial mycelium, non-chromogenic type, no or weak hydrolysis of starch and proteolytic action.

Among the known species of Streptomyces, S. verticillus, S. Cinnamoneus and S. flavopersicm are known to be the whorl-forming and non-chromogenic type. In the next table, the strain B80-Z2 and the known species are compared.

Strains Properties BSD-Z2 S. vert- S. cinn- S. flavicillus amoneus opersicus Whorl Spiral Chromogenecity Hydrolysis of Starch 1- 1 4+ Nitrate Reduction Proteolysis -ll- -H Color of grayish with grayish cinnamon pale aerial mycelium olive tinge olive yellowish on synthetic with with med. pinkish pinkish tinge tinge Color of colorless, cream cream yellowish growth yellowish to color color brown Antibiotic Bleomycin Phleo- Cinnamycin Actinproduced mycin Duramycin ospect- Heptaene acin As shown in the above table, they are very similar to each other except for minor differences. Among the above species, the strain B80-Z2 resembles most S. verticillus, while S. cinnamoneus possesses cinnamoncolored aerial mycelium, strong hydrolysis of starch and strong proteolytic action, which are not shown by the strain B80-Z2. S. flavopersicus forms a pale yellowish aerial mycelium on the synthetic agar and shows significant proteolytic action in the skimmed milk and the gelatin medium, by which the strain B80-Z2 is differentiated. in addition, the strain B80-Z2 grows very well in the medium consisting of starch as the carbon source and soybean flour as the nitrogen source and it is suggested that hydrolysis of starch and proteolysis can be varied depending on cultural conditions. Therefore, the differences between the strain B80-Z2 and S. verticillus in respect of starch hydrolysis and proteolytic action is not considered to be significant. S. verricillus forms usually pinkish-gray aerial mycelium, but occasionally olive gray aerial mycelium, which is very much similar to that of the strain B80-Z2. In other respects, such as nitrate reduction, there are differences between them at some extent, but the majority of their characteristics are common between them and it is considered to be proper to conclude that the strain B-Z2 belongs to S. verticillus.

It is generally recognized that actinomycetes are easily mutated. This invention is not to be limited to the production of bleomycin by S. verticillus, the strain 1380-22 or organisms fully answering the above description. S. verticillus in this patent application includes the strain B80-Z2 and its artificial and natural mutants. In other words, in this patent application all organisms which produce bleomycin are included in S. verticillus, except those which are differentiated from S. verticillus without any ambiguity or any uncertainty.

When S. verticillus is cultivated under proper conditions, bleomycin is produced. Mycelia or spores of a bleomycinproducing organism are inoculated to a proper medium and fermented aerobically to yield the cultural product containing bleomycin. Bleomycin production can be achieved by the cultivation on a solid medium but it is recommended that it be cultivated in a liquid medium for large-scale production. The incubation temperature may be any temperature at which a bleomycin-producing strain is able to grow, but it is preferable to conduct the fermentation at 25-35 C., especially at 27-32 C. The medium for the production of bleomycin consists of nitrogen sources, carbon sources, inorganic salts with or without stimulatory factors for production and others. Carbohydrates, fats or oils can be used as the carbon source. For instance, starch, glucose, glycerol, maltose, dextrin and sucrose are used in either purified or crude state. As nitrogen sources, soybean flour, meat extract, distillers solubles, peanut flour, peptone, fish meal, yeast extract, com steep liquor, nitrate, ammonium salts, urea, etc. are used. Inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, phosphates etc. are added if necessary. Heavy metal salts such as copper, manganese, iron, zinc, etc. are added to the medium if necessary. All materials which are known to be used for the cultivation of actinomycetes, such as those indicated in Canadian Pat. No. 513,324, British Pat. Nos. 730,341, 736,325, US. Pat. Nos. 2,691,618, 2,658,018, 2,653,899, 2,586,762, 2,516,080, 2,483,892, 2,609,329, and 2,709,672 are available for the production of bleomycin. To prevent foaming during the fermentation, all known antifoaming agents such as parafiin, fat, soybean oil, silicone resin are utilized. All other known methods for the fermentation of penicillin, streptomycin or for the production of other antibiotics can be employed for the production by fermentation of bleomycin.

Experimental methods employed in this invention are as follows unless otherwise noted specifically:

l Shaking culture:

The medium cc.) was placed in a Sakaguchi flask of 500 cc. volume and sterilized at C. for 20 minutes. Mycelia, spores or their mixture of the bleomycin-producing strain were inoculated into the above sterilized medium and cultured on a shaking machine (120 strokes per minute with 8 cm. amplitude) at 2729 C. One ml. of second days broth was inoculated as the inoculum into 100 cc. of a medium in a flask of 500 cc. volume and cultured.

2. Tank culture:

volume: A tank was charged with 180 liters of a medium and sterilized at 120 C. for 30 minutes. Aeration was 200 liters per minute and the stirring was 200 rpm. The temperature was kept at 2720 C. Soybean oil or silicone resin was employed as the antifoam.

3. Assay of bleomycin:

Mycobacterium phlei NIHJ was used as the test organism and the cylinder plate method was employed.

the cylinder plate assay method for penicillin. A lot of bleomycin which was extracted from culturefiltrate by the ion exchange resin process followed by Sephadex chromatography was designated as the standard. The activity was expressed by the weight of the standard.

The bleomycin-producing strain was first shake-cultured by the inventors in the following medium. The medium consisted of 1.0 percent glucose, 1.0 percent starch, 0.75 percent meat extract, 0.75 percent peptone and 0.3 percent sodium chloride. The initial pH was adjusted to 7.0. Then at 4 days culture the pH of the broth was 7.3. This broth showed inhibition zone of mm. in the cylinder plate method.

Bleomycin production was examined in media consisting of various kinds of carbon and nitrogen sources by the shaking culture. The results of production of bleomycin and the pH of cultured broth are shown in the following table.

(I).-TEST OF CARBON SOURCES [Basal medium consists of 0.75% meat extract, 0.75% peptone, 0.3% NaCl] Days 2nd 3rd 4th 5th Medium Carbon Number sources p11 McgJml. pH Meg/ml. pH Meg/ml. pH McgJml. 1 7. s 0. 25 (a. 2 0. 37 7. 4 0. as 7. s 0. as 2. 1 2% glucOStL 7. 0 0. 20 (5. B 0. 34 7. 4 0. 38 7. 0 O. 37 3 2% starcln 6. 4 0. 07 5. 4 0. l0 7. 0 0. 36 7. 6 0. 47 1 2% lactose 8. 2 0. 27 8. 0 0. 26 8. (i 0. 32 8. 8 0. 11 5 2% sucrose. 8. 0 0. 32 8. 2 0. 17 8. 2 0. 17 8. 2 0. 14 G 2%1nalt0se" 8. 2 0. 20 0. 6 0. 10 4. x 0.37 5.0 0.37 7 2% dextrln 5. 8 0. 05 0. 0 0. 7. 2 0. 44 7. 0 0. 38 b 2% glycerol" 0. 8 0 ll. 4 0 0. 2 0. 07 0. 4 0. 18

(ll).-'lEST 0F NITROGEN S0 URGES [Basal medium consisted 01 1% glucose, 1% starch and 0. 3% NaCl] Days 2nd 3rd 4th 5m em Medium Nitrogen sources and Mcg./ Mcg./ Mcg./ MegJ Mcg./ Number salts pH ml. m1. ml. m1. m1.

1 i ii f j 6. 4 0.27 7. 0 1. 23 7. 2 1. 41 7. 4 0.69 s. 0 0. 24

1.5% soybean flour 2 0.1% K HPO 6.4 0 6.4 0.06 6.0 0.41 7.0 1.40 7.8 0.90

0 05% MgSO4. 1% soybean flo a 8' j g 3- e. s 0. 03 7. z 0. 0e 7. o 0. 3s 7. 4 0. 45 8. 6 0. 29

0.5% corn steep liquor 0.74% meat extract- 4 0.75% peptone 7. 2 0. 25 7. 0 1. 35 7. 0 1. 00 8. 2 0. 43 8. 2 0. 39

0.3% yeast 5 2% corn steep liquor 6.8 0 6. 8 0.06 6. 8 0. 22 6. 8 0. 06 7. 2 0. 06 6 m 3- 6.6 0 6.6 0. 13 e. 4 0. 31 7. 2 1. 19 8.2 0.20 7 7. 0 0. 09 0.6 0. 79 7. 2 1. 71 7. 4 1. 47 8.0 1. 44

In Medium No. 7, starch was present at 2.0%.

Mycobacterium phlei was shake-cultured in glycerol bouillon at 27-29 C. for one week and was used as the test organism. The medium for the cylinder plate method consisted of 1.0 percent glycerol, 1.0 percent meat extract, 1.0 percent peptone, 0.2% NaCl and 2.0 percent agar. Five m1. of 1.5 percent malachite green solution was added to 1,000 ml. of the medium. The pH was adjusted to 7.0. After the sterilization at 120 C. for 20 minutes, to this medium of 95 cc., 5 cc. of M. phlei culture was added and used for the seed layer. The other procedures were the same as those employed in The above results are examples of tests. Starch, dextrin, glucose, lactose and maltose are examples of carbon sources useful for the production of bleomycin.

cent glucose, 3.5 percent soybean meal, 0.1% K I-WO 0.05 percent zinc sulfate and 0.01% CuSO, H O (pH 7.0) is an example of media useful for the production of bleomycin.

A strain which was selected from the original culture of the strain No. B80-Z2 by the single colony selection was shake-cultured in the above medium and yielded 100 meg/ml. of bleomycin after the 7-10 days shaking of the culture. At the time of maximum production the pH of the cultured broth was 8.0-8.2. It is generally known that compositions of high yielding media for the production of an antibiotic vary depending on strains, even in cases in which they derived from the same original strain. It is the same in the case of the bleomycin-producing strains. The media compositions most suitable for the bleomycin production must be varied depending on strains obtained by monospore-selection or by the treatment of the ultraviolet irradiation or others. High production of bleomycin can be achieved by known procedures, for instance, by the irnprovement of the strain by monospore-selection, by treatment with ultraviolet light, X-ray or other mutagens and by the selection of media suitable for the bleomycin production by each improved strain.

Bleomycin exists mainly in the liquid part of the fermented broth. The liquid part containing bleomycin is separated from the solid mass in the cultured broth by known procedures such as filtration or centrifugation. As described later, bleomycin is adsorbed on an ion exchange resin. After the removal of the solid mass from the cultured broth, bleomycin is adsorbed on the resin. However, the ion exchange resin process can also be applied for adsorption of bleomycin directly from the cultured broth after removal of large particles in' the broth by a proper screen. Bleomycin in the cultured broth or in the filtrate is stable enough for evaporation in vacuo or spray drying. The dried material thus obtained can be applied for the agricultural use.

Bleomycin is not practically transferred from the aqueous solution to solvents such as butanol, ethyl acetate, ether or benzene. Solvent extraction can be utilized for elimination of some impurities, if desired.

An aqueous solution containing bleomycin can be concentrated by distillation in vacuo and dried. During this procedure, it is preferable to keep the pH at 6-7, since bleomycin is most stable in this pH range. Washing of the dried material with solvents such as acetone, butanol, ethanol or ethyl acetate in which bleomycin is practically insoluble is useful to remove impurities soluble in the used solvents. Bleomycin in the dried material can be dissolved in water or methanol. Bleomycin dissolved in water or methanol can be precipitated with solvents in which bleomycin is insoluble and which are miscible with water or methanol.

Bleomycin in aqueous solution or in cultured broth is adsorbed on activated carbon and is eluted from the carbon at acid pH by aqueous methanol, aqueous ethanol, aqueous acetone or water-saturated butanol. The yield of bleomycin in this procedure was usually less than that obtained by an ion exchange resin process which is described below.

Bleomycin is basic and water-soluable, therefore, an ion exchange resin process can be employed for extraction and purification of bleomycin in high yield. lon exchange resins which have carboxyl or phenol radicals are able to adsorb bleomycin on the basis of its basic nature and on the basis of Van der Waals power. Cation exchange resins of the carboxyl type and carboxymethyl cellulose are suitable adsorbents for extraction and purification of bleomycin. For an example, bleomycin is adsorbed on a cation exchange resin such as Amberlite [RC-50 (product of Rohm and Haas Co.). The use of this resin in H form is more preferable than Na form. This resin is placed. in a column and is converted to H form. Then an aqueous solution containing bleomycin, e.g., culture filtrate adjusted to pH 4 6.5, is passed through the column. Thereafter the column is washed with water. Then elution is made with dilute hydrochloric acid. The eluate of bleomycin, thus obtained, is adjusted to pH 5-7 and is lyophilized or dried in vacuo. Bleomycin in the eluate can be precipitated by addition of solvents, for instance, ten volumes of acetone, in which bleomycin is insoluble, and the precipitate is dried.

Bleomycin in the dried powder is dissolved in methanol. From the methanol solution bleomycin can be precipitated by addition of acetone, ether, etc. Washing of the powder containing bleomycin by ethanol is useful to remove impurities.

Depending on the molecular size of bleomycin, methods of separating substances on the basis of molecular sieves, such as those using Sephadex, is useful for the purification of bleomycin. A bleomycin aqueous solution is applied on Sephadex g-25 column. The latter is a commercially available, cross-linked dextran polymer which forms a gel with water and acts as a molecular sieve, absorbing molecules smaller than about 3,000 molecular weight. The active eluate is concentrated in vacuo or lyophilized. Thus, bleomycin is purified. By Sephadex column chromatography bleomycin was separated into bleomycin A having Rf value 0.94-0.88 and bleomycin B having Rf 0.70-0.66. This Rf value was shown by paper chromatography using 10 percent ammonium chloride. Other than these two main components of bleomycin, two or three antibacterial substances were shown to be present in a very small quantity. The purified bleomycin A and B, thus obtained, is white or white with pale green to bluish tinge.

When tested by paper chromatography using 10 percent ammonium chloride, the cultured broth and the extract obtained by the process using Amberlite lRC-50 from the cultured broth, showing usually two large spots and two or three small spots which have antibacterial activity against Mycabacterium phlei. Bleomycin in this application means each of the substances shown in the two main spots or the mixture thereof. In other words, the mixture of these two main substances or each of those is called bleomycin, unless otherwise noted. Among these substances, the substance of Rf 0.94-0.88 is designated bleomycin A and that of Rf 0.70-0.66 bleomycin B. Except for the differences of Rf values, bleomycin A and B are very similar to each other.

The properties of bleomycin are as follows: Bleomycin is pale greenish or bluish white powder. A clear melting point is not observed, but the separated powder of bleomycin A and bleomycin B decomposed at over 106 C.

Bleomycin and its hydrochloride are soluble in water, soluble in methanol, slightly soluble in ethanol, and hardly soluble in acetone, ethyl acetate, butyl acetate and ether.

Bleomycin is adsorbed on a cation exchange resin and eluted with aqueous inorganic acids.

The absorption spectra of bleomycin A and B are shown in FIG. 1, indicating absorption at 243-244 my and maximum at 290-295 ma.

The infrared absorption spectra of bleomycin A and B in KBr tablets are shown in FIG. 2, representing maximum absorptions at wave numbers (cm) of 3380-3220, 2940, 1715 (sh), 1650, 1635- 1605, 1560-1540, 1510, 1450-1440, 1380-1365, 1250, 1090-0145 and 1010 (sh).

Bleornycin gives positive Pauly, Ehrlich, Dragendorf and permanganate tests but gives negative Molisch, biuret, Elson-Morgan and maltol reactions. As described below, bleomycin A is further separated to six components, all of which give a negative Sakaguchi test. Bleornycin Al, A2 and A3 give negative ninhydrin tests and A4, A5 and A6 give positive ninhydrin tests. Bleornycin B is separated into five components and all give negative ninhydrin and positive Sakaguchi reactions. Thus, bleomycin, bleomycin A and bleomycin B give positive or negative ninhydrin and Sakaguchi reactions, depending on the amount of each component.

On the paper chromatogram developed by 10 percent ammonium chloride, bleomycin appeared at Rf 0.94-0.88 and bleomycin B at Rf 0.70 0.66. On the paper chromatogram developed by n-propanol: pyridinecacetic acidzwater (:10:3zl2), bleomycin A is at Rf 0.50-0.42 and bleomycin B is at Rf0.570.54. ln paper chromatography using water-saturated n-butanol containing 2 percent p-toluene sulfonic acid, both bleomycin A and B fail to move from Rf 0 even after 72 hours development.

The aqueous solution of bleomycin absorbed D line, and it was hard to determine its optical rotation.

Since bleomycin is not obtained in the crystalline state, it is hard to determine the molecular formula of bleomycin until the structure will be determined.-

Moreover, it is hard to reach complete purification of bleomycin A and B. A sample of the hydrochloride of bleomycin B was colored pale blue and it was found to contain metals by fluorescent X-ray analysis. Cu, F and K were shown in the ratio 28:8:0.05. Accordingly, bleomycin B contained 0.27 percent of Cu, if all metals contained in bleomycin B were calculated as Cu. In the case of bleomycin A, the value of Cu was calculated to be 0.08 percent. In the course of purification of bleomycin, it was impossible to separate the greenish color from bleomycin and bleomycin was shown to have a property of chelating with copper. But a sample of purifled bleomycin A contained almost no copper.

Bleornycin forms acid addition salts such as hydrochloride, sulfate and its reineckate and helianthate precipitate.

Bleornycin A and B have antimicrobial activity against various bacteria and fungi, including plant pathogens. The activities shown by the agar streak method are as follows:

Escherichia coli Nll-U 3. l25-1.6 0.8-0.4 E. coli (streptomycin fast) 3.l25-1.6 1.6-0.8 E. coli (neomycin fast) 3.l25-l.6 1.6-0.8 E. coli (chloramphenicol fast) 1.6-0.8 0.8-0.4 E. ooli (kanamycin fast) 3. l25-1.6 1.6-0.8 E. coli (streptomycin fast) 3. 125-1 .6 1.6-0.8 Klebsiella pneumoniae (PCI 602) 3.l251.6 1.6-0.8 Salmonella typhi 63 3. l25-l.6 1.6 Salmonella typhimurium 1406 1.6-0.8 0.4-0.2 Salmonella paralyphi A1015 1.6-0.8 1.6 Salmonella paratyphi B8006 1.6-0.8 3.125-1 .6 Salmonella pamtyphi C Hirschfeld S-33 0.8 0.2 Salmonella cholerac suis I348 0.8-0.4 0.4-0.2 Shigella dysemeriae 2-1684 0.2 0.4-0.2 Shigella dyremeriae Ohara 50-25 12.5 Shigellaflexneri la-ll 0.2 0.4-0.2 Shigella boydii l-65 1.6-0.8 0.8-0.4 Shigella sonnei l-l 196 1.6-0.8 0.4 Pmteur vulgar-is 0X-19 0.4 0.2 Pseudomonas pyogenes A3 100 Corynebaclerium xemtris 0.1-0.05 0.2-0.1 Mycobacterium phlei 0.0004- 0.004-

0.0002 0.0002 Mycobacterium 607 0.2 0.4 Mycobacterium (streptomycin fast) 0.1-0.05 0.1 Mycobacterium (kanamycin fast) 0.1-0.05 0.2-0.1 Cladosporium sphaerrperum $0 100 Calleclrichum phomoides 25 50 Carticium centrifugus 1.6 3.2 Fusarium lini 25 100 Fusarium oxysparum 50 100 Furarium mreum 1.6 6.2 Gibberella fitjikuroi 100 100 Gibberella saubinetii 100 100 Gloeosponum kaki 0.8 1.6 Glomerella Iaginarium 1.6 6.2 Helminlhosporium seanum 0.4 0.8 Oiriculan'a gn'sea 50 100 Piricularia oryzae 100 100 Selerotium roefrii 0.2 1.8 Pseudomonas rolanacearum 100 100 Xanthomanas oryzae 0.4 0.8 Asperigillus niger 100 100 Candida albicans 3417 100 100 Candida albicans YU-l 200 100 100 Cryptococcus neoformans 7496 6.2 100 Histoplasma caplrulatum 4206 0.4 1.6 Trichophyton mentagrophytes 640 100 100 Botritis bassiana 100 100 Sacchammyces cerviriae l 1299 25 100 Saccharomyces cervisiae petit. 12.5 25

The toxicity of bleomycin was low and the intravenous injection of 8 mg./mouse did not cause death.

Bleornycin inhibited experirnentalanimal tumors, such as sarcoma 180, Ehrlich tumor in mice, by the daily injection of 25 meg/mouse for 10 days. HeLa cells in tissue culture was inhibited at 25-1 2.5 mcg./cc. but Yoshida sarcoma cell in tissue culture was not inhibited at 10 mcgJcc. Bleornycin A showed stronger activity than B to Ehrlich carcinoma and sarcoma 180.

In order to confirm bleomycin to be a new substance, it is necessary to compare bleomycin with known antibiotics as follows. Among known antibiotics inhibiting Gram-positive and Gram-negative bacteria, streptomycin, kanamycin and paromomycin are easily differentiated from bleomycin, in respect of the paper chromatograph using water-saturated butanol containing p-toluene sulfonic acid, inhibition of their resistant organisms, and the antibacterial spectrum. Since bleomycin is produced by the whorl-forming streptomyces, it is necessary to compare bleomycin with actinospectacin or phleomycin which are produced by streptomycetes forming whorls. Actinospectacin is differentiated by the fact that actinospectacin inhibits Staphylococci at 10-100 mcg./cc. and it is not inhibito ry against sarcoma 180 of mice, being difierent from bleomycin.

Phleomycin, which was discovered by the present inventors, has inhibitory activity against Gram-positive and Gram-negative bacteria, Ehrlich carcinoma and sarcoma 180 of mice, and the strain producing phleomycin has many common characters with the bleomycin-producing strain. Accordingly, it is necessary to compare bleomycin carefully with phleomycin. When their antibacterial activities were compared with each other, significant differences revealed were as follows:

Bleomycin can be differentiated from phleomycin also in respect of stability in acid and alkaline state. Thus aqueous solutions of phleomycin and bleomycins A and B at 100 meg/cc. were adjusted to pH 2 and 9 and were kept at room temperature. After storage for the specified period of time a part of each solution was taken out and measured for activity against Mycobacterium phlei. The result is shown in the following table; bleomycin is significantly more stable at acid and a1- kaline pl-l than phleomycin.

Remained Activity (36) After Storage of 15 30 60 I20 240 pH Substances min. min.min. min. min. min. BleomycinA 100 100 87 85 59 53 2 BleomycinB 100 100 80 84 63 46 Phleomycin 89 85 53 39 21 20 Bleomycin A 100 100 74 65 48 20 9 BleomycinB 78 61 49 37 33 2O Phleomycin 8 8 8 8 2 1 Bleomycin and phleomycin can be differentiated also on paper chromatography developed with 10 percent ammonium chloride. That is, bleomycin A gives Rf 0.94-0.88, whereas phleomycin shows two to six spots at Rf below 0.81. Bleomycin B is not difl'erentiated from phleomycin by paper chromatography but they can be differentiated by the difference in their absorption of ultraviolet light. Phleomycin shows two absorption maxima at 244-246 and 301 mu, while bleomycin B shows absorption at 243-244 mp. and maximum absorption at 291-295 mu of which E is larger than that of phleomycin at 301 mu.

Properties of bleomycin, bleomycin A and bleomycin B which are obtained from the cultured broth by the ion exchange resin and Sephadex G 25 chromatography were described and they were enough to differentiate bleomycin from known antibiotics. Thin layer chromatography of bleomycin using Silica gel G and 10 percent ammonium acetate-methanol (1:1) shows A1, A2, A3, A4, A5, A6 in bleomycin A and B1, B2, B3, B4, B5 in bleomycin B. Bleomycin A and B have a property of chelating with copper and they were more easily purified in their copper-containing forms. Therefore, the further purification, that is, the separation to each of their components, was made in their copper-containing forms. The alumina chromatography was also a helpful process for the purification, and, though the occurrence of an increase in toxicity was suggested to be caused by this process, in order to clarify the physico-chemical property of each component of bleomycin, alumina chromatography was used. When bleomycin was dissolved in methanol and treated by alumina chromatography using methanol, bleomycin increased in toxicity to mice (three to four times) and the therapeutic index of bleomycin thus treated became three to four times smaller than bleomycin before the treatment. Therefore, a slight structural change was suggested to occur during this process. For convenience, bleomycin A and B treated by the alumina chromatography were designated bleomycin At and Bt respectively. The copper-containing At and Bt were designated Cu-At and Cu-Bt and they were obtained by addition of copper chloride. Cu-At and CuBt were further purified by gradient column chromatography using CM Sephadex C25 and aqueous ammonium formate, the concentration of which was raised from 0.1 M to 1.0 M. Cu-At were separated to six active components designated Cu- Atl, Cu-At2, Cu-At3, Cu-At4, Cu-AtS, and CuAt6. Cu-Bt were separated to 5 components designated Cu-Btl, Cu-Bt2, Cu-Bt3, Cu-Bt4 and CuBt5. Generally, Cu-At2 and Cu-At5 were the main components of Cu-At, and Cu-Bt2 and Cu- Bt4 were the main components of Cu-Bt. The concentrations of ammonium formate where each of these components was eluted, Rf of each component by the paper chromatography using 10% Nl-LCl (PPC), Rf values on the thin layer chromatography using Silica gel G and 10 percent ammonium acetate-methanol (1:1) (TLC), and relative mobility (Rm) by the high voltage electrophoresis using formic acid-acetic acidwater (25:75:900 in volume, pH 1.8) under 2000V and 25 mA (l-lVE) are shown in the following table. Rm values were calculated, taking L-alanine as the standard (Rm 1.0).

in the paper chromatography using 10 percent ammonium chloride, all Cu-At of bleomycin showed Rf values of 0.83-0.92 and all Cu-Bt showed Rf values of -0. 72. All CuAt and Cu-Bt showed positive Pauly, Ehrlich, Dragendorf and permanganate reactions. Cu-At1, Cu-At2 and Cu-At3 gave negative ninhydrin. CuAt4, CuAtS and CuAt6 gave positive ninhydrin. All CuBt gave negative ninhydrin. All CuAt gave negative Sakaguchi and all CuBt gave positive Sakaguchi reactions. All CuAt and Cu-Ht gave negative Tollens, Ferric chloride, Fehling and Molisch reactions. All CuAt and Cu-Bt showed a similar type of ultraviolet spectra having maxima at 244 my. and at 295 mp. and the spectra of CuAt2 and CuBt2 are shown in FIG. 3. The infrared spectra of CuAt2 and CuBt2 are shown in FIG. 4. The elemental analysis of CuAtZ showed the following result: Cu, 5.41. CuAt and CuBt gave the copperfree form by the treatment with S-hydroxy-quinoline or with dithizone at acid pH. Comparing the antitumor activities, CuAt2 and CuAtS showed a higher therapeutic index than CuBt2 and Cu-Bt4. Cu At showed the highest therapeutic index.

A more definite comparison of bleomycin with phleomycin can be made by the comparison of each component of bleomycin with each component of phleomycin. A blue powder of phleomycin containing copper at 4.38 percent which was obtained by the ion exchange resin process followed by alumina chromatography was further purified by step-wise elution from CM Sephadex C25 column using ammonium formate (1-5 percent), that is, principally by the same process used for bleomycin. Then phleomycin was separated into phleomycins A, B, C, D1, D2, E, F, G, H, l. The concentrations of ammonium formate at which each component was eluted was as follows: A and B 0.79M, C, 0.16M, D1 and D2, 0.24M, E, 0.32M, F, 0.395M, G, 0.47M, l-l, 0.63M, l, 0.71M. All phleomycins were differentiated from all bleomycin CuAt by paper chromatography using NH Cl and by Sakaguchi reaction. All phleomycins showed Rf below 0.80 and gave positive Sakaguchi reaction. All phleomycin except C, D2, F had maxima at 244 my. (E 120-140 and 301 m (E 40-50) and were differentiated from all bleomycin CuAt and CuBt. Phleomycin C, D2 and F had maxima at 244 mp. (E 120-145) and 295 m;.t(E 90-115), and this ultraviolet spectrum was similar to those of CuAt and CuBt of bleomycin. Therefore, C, D2 and F were carefully compared with Cu-Bt group of bleomycin. Then, bleomycin CuBt2 and CuBt5 were not differentiated from phleomycin C and F, respectively. Others were differentiated by the thin layer chromatog raphy. Phleomycins gave positive Ehrlich, Dragendorf and Sakaguchi, but negative ninhydrin tests. All phleomycins and bleomycins gave a positive test for sulfur.

Bleomycin is promising to be useful for therapy against infectious bacterial diseases of human, animal or plant.

Examples of processes for production and isolation of bleomycin are described below. It is apparent to specialists who are skilled in this field that the variation or modification of the processes here described can be easily made on the basis of cultural properties which are known in streptomycetes and on the basis of the properties of bleomycin above described.

The next examples are given merely for illustrative purposes only and the invention is not limited to those examples.

The strain B-Z2 was shake-cultured in medium consisting of 1 percent glucose, 1 percent starch, 0.75 percent meat extract, 0.75 percent peptone and 0.3% NaCl (the initial pH 7.0) at 27-28 C. for 5 days. Mycelium mass was removed from the cultured broth by filtration and the filtrate of 3.4 liters contained 1.23 mg. of bleomycin. The filtrate pH 7.8) was adjusted to pH 6.4 and passed through the column of 300 cc. of lRC-SO (H type) (2.8 cm. in diameter). The flow rate was 10 cc. per minute. After passing of the broth filtrate, the column was washed with distilled water. The effluent and the wash water showed no potency, suggesting bleomycin adsorption. This column was eluted with 0.2 N l-lCl at flow rate of 5 cc. per minute and the eluate was fractionated to each 50 cc. The fractions up to 12th had pH 5.4 -4.8, and did not contain bleomycin. At the 13th fraction and thereafter, the pH decreased to 2.0. At the 14th fraction, the pH value decreased to below 1. Bleomycin was contained in 450 cc. of 13th 21st fractions. These fractions were neutralized with Amberlite lR-4B (0H type) immediately after the elution. The active fractions were combined and lyophilized. The lyophilized powder was extracted with methanol (30 cc.) and separated from the residue. The residue was extracted twice with 10 cc. of methanol. These methanol extracts were combined and dropped into ethyl ether (500 cc.). The precipitate occurring was collected by centrifugation and dried in vacuo at room temperature, yielding a brownish colored powder of 1.808 g. This powder con.- tained 1.2 mg. of bleomycin and 1 mg. of this powder contained 0.67 mcg. of bleomycin.

EXAMPLE 2 From the powder of bleomycin hydrochloride which was obtained in Example 1 and which had the activity of 0.67 meg/mg. 1.7 g. was taken and extracted with ethanol of 25 cc. Ethanol-insoluble powder was dissolved in methanol, and this methanol solution was passed through the column (1.3 cm. in diameter) of 5 g. alumina (Grade 1). From this column, elution was made by methanol, 10 percent aqueous methanol and 20 percent aqueous methanol successively. Bleomycin was eluted in absolute methanol and 10 percent aqueous methanol. This bleomycin-containing eluate was concentrated in vacuo and dried, yielding 550 mg. of brownish-colored powder containing bleomycin hydrochloride at 1.7 meg/mg.

EXAMPLE 3 Each 10 liters of a medium consisting of 3.5 percent soybean flour, 2.5 percent starch, 0.5 percent glucose, 0.3% NaCl, 0.1% K HPO 0.05 percent zinc sulfate and 0.01% CuSO (the initial pH 7.0) was placed in two stainless steel jar fermentors (30 liters in volume) and sterilized. This medium was inoculated with 600 cc. of the shaking-cultured broth of the strain B80-Z2 in the same medium for 2 days. Fermentation proceeded under aeration of 10 l. per minute and stirring at 500 rpm. at 2729 C. Silicone oil was added at 0.0033 percent as the antifoaming agent to the medium before the start of the fermentation and during the fermentation a total of 50 cc. of soybean oil was added. The

broth was sampled (10 cc.) at various days during the fermentation and the pH and the content of bleomycin were found to be as follows:

Days

Fermentor 2 3 4 5 6 8 9 10 pH 5.8 5.8 6.4 6.4 6.2 5.4 5.7 No. l

McgJcc. 4.5 5.8 18.2 80.0 pH 5.8 5.3 5.4 5.8 5.0 5.0 5.8 6.0 No.2

Meg/cc. 4.5 5.1 13.7 29.5

Bleomycin Fraction Volume content No. cc. pH Meg/cc. Mg.

The fractions of No. 3-6 were combined and neutralized by Amberlite lR-4B (OH type). The neutral eluate was concentrated in vacuo. The concentrated solution was washed with acetone and dried in vacuo. The brownish powder thus obtained was extracted with methanol (150 cc.). The methanol-insoluble part (11.59 g.) contained 57.5 mg. of bleomycin (4.98 mcg./mg.). The methanol solution was distilled and dried to powder. This powder was extracted with ethanol of 100 cc. The ethanol-insoluble powder (4.038 g.) contained 293 mg. of bleomycin (72.5 mcg./mg.). This grayish powder was dissolved in distilled water (5 cc.) and applied to the column of Sephadex G-25 (200 cc., 2.0 cm. in diameter). Thereafter elution was made by distilled water. The effluent was cut into fractions of 5 cc. each and each fraction was lyophilized.

Effluent Lyophilized Powder Con- Con- Fraction Bleomycin tent Wt. Bleomycin tent No. Color McgJcc. Mg. Mg. Meg/Mg. Mg.

1-10 Colorless 0 0 11-13 Pale Brown 62.3 0.24 0.15 14 Pale Brown 29.5 5.6 0.17 15 Pale Brown 0.02 0.11 36.4 3.49 0.13 16 Pale Brown 0.084 0.42 45.1 2.84 0.13 17 PaleBrown 0.184 0.92 53.0 16.50 0.88 18 Pale Brown 0.256 1.26 30.6 70.00 2.14 19 Brown 0.555 2.78 68.0 76.00 5.15 20 Brown 0.21 1.53 115.0 30.60 3.53 21 Brown 021 1.04 86.2 21.35 1.83 22 Greenish 12.9 64.5 [604275.00 44.

Brown 23 Green 14.0 69.5 23333900 49.5

24 Green 1 1.4 57.0 244.6310.00 76.0 25 Green 8.05 40.3 249.5332.00 82.5 26 Pale Yellow- 6.23 31.3 270519200 52.0

ish Green 27 Pale Yellow- 1.56 7.75 331.9 51.00 26.9

ish Green 28 Pale Yellow 0.78 3.91 371.4 12.20 4.60 29-32 Pale Yellow 029 5.75 1 192.5

The above fractions containing bleomycin were paper chromatographed using Toyo filter paper No. 51 developed with 10 percent ammonium chloride solution by ascending method. After development for 3-4 hours, the solvent ran about 30 cm. and the filter paper was dried in air. Bioautographs of the paper chromatograms were made against Mycobacterium phlei. As to results, fractions No. 13-15 showed a small inhibition zone at Rf 0.51-9.57, fractions No. 16-23 showed a large inhibition zone at 0.94-0.88 (bleomycin A), and No. 24-29 at 0.70-0.66 (bleomycin B). In the fractions after the 30th, another substance showing a partial inhibition at RfO.83 was detected besides bleomycin B at Rf0.70-0.66.

EXAMPLE 4 The fraction No. 24 of Sephadex G-25 chromatography in Example 3 gave 244.6 mg. of bleomycin powder (containing '76 mg. of bleomycin, 310 mcg./mg.) and this powder was dissolved in 20 cc. of warm methanol. After cooling, the insoluble powder of 35.5 mg. (containing 1.49 mg. of bleomycin, 42 meg/mg.) was filtered off, and to the methanol solution ethanol of 20 cc. was added to result in a finely powdered precipitate. This powder of 46.9 mg. (containing 1.87 mg. of bleomycin, 39.8 meg/mg.) was removed by filtration and the filtrate did not produce any more precipitate by addition of ml. ethanol. This solution was distilled in vacuo and yielded a practically colorless precipitate of bleomycin. After concentration to 20 cc. volume, it was kept at 0-5 C. for 6 hours and pale bluish powder mg.) was obtained after filtration and drying. This powder decomposed at 192196 C. on microblock and contained 742.5 mcg. of bleomycin hydrochloride per one mgm. By paper chromatography developed with ammonium chloride, this powder was shown to be bleomycin B.

On the other hand, mg. of fraction No. 22 (containing 27.5 mg. of bleomycin, 275 meg/mg.) was treated in the same way as above and finally a purified white powder 27.9 mg.) was obtained.

EXAMPLE 5 Powder (633 mg.) containing 0.65 mg. (1.03 meg/mg.) bleomycin was applied to a column 1 cm. in diameter) of cellulose powder (10 g.) which was treated with a mixture of n-propanol-pyridine-acetic acid-water (15:10:32 in volume ratio). The partition chromatography was made by the same solvent mixture. All bleomycin was found in fractions No. 6-16 when the efiluent was fractionated by 5 cc. These active fractions were collected and concentrated to about 5 cc. volume. Ethyl ether (50 cc.) was added to this 5 cc. to yield a precipitate of bleomycin. After drying this precipitate it was washed with ethanol and the methanol-insoluble part was removed. Tl-le methanolsoluble part was dropped into ethyl ether 10 volumes) and yielded a powder weighing 36.27 mg. and containing 0.62 mg. of bleomycin 1.7 meg/mg.

A brown powder (1980 g.) showing an activity of 3 rig/mg. which was obtained by the [RC-50 resin process from culture filtrate, was dissolved in methanol at the rate of 4 cc./g. The insoluble part was discarded. The methanol solution was concentrated in vacuo at 2030 C. almost to dryness, and ethanol was added at the rate of 2 cc./g. Ethanol was removed. The dried powder thus obtained contained bleomycin, and this powder was further treated twice by the same procedure, that is, dissolution in methanol (4 cc./g.), concentration in vacuo and addition of ethanol (2 cc./g.). Then, 58.1 g. of a brownish powder showing an activity of 93 rig/mg. was obtained. Thirty-four g. of this powder was dissolved in 170 cc. of methanol and to this methanol solution 27.2 cc. of percent copper chloride in methanol was added. This amount of copper chloride solution was determined by testing the necessary amount for the chelation which was determined by the optical density at 620 mg" The solution was kept for 30 minutes at room temperature and filtered. The filtrate was applied to the alumina column (340 g. of alumina in methanol). Methanol was further passed and the eluate obtained from the blue ring was evaporated in vacuo, yielding 7.9 g. of blue powder containing bleomycin CuAt and CuBt. 1f the activity is shown by the comparison with the working standard of bleomycin, it showed 215 p. g/mg. Four g. of this powder was dissolved in distilled water and the solution was applied to Sephadex G- column (4 X 80 cm.). The flow rate was 0.5 cc./m. When the blue band on the column came down, the blue eluate was cut into three parts depending on the results of the paper chromatographic analysis. From the first part, 517 mg. of the blue powder showing an activity of 506 ug/mg. was obtained. It was shown to contain bleomycin CuAt (Rf 0.85-0.95) by paper chromatography using 10% NH C1. The second part gave a blue powder of 166 mg. showing an activity of 520 ug/mg. and was shown to contain CuAt and CuBt. The third part gave a blue powder of 3154 mg. showing an activity of 111.6 pig/mg. and was shown to contain CuBt (Rf 0.70-0.75) by a similar procedure from another culture filtrate, a blue powder of CuAt, 1.903 g. was ob tained, and it showed an activity of 269 rig/mg. This powder was dissolved in 0.1M ammonium formate (pl-l 6.4) and applied to CM Sephadex C-25 column (2 X 35 cm.). Gradient elution was carried out by increasing the concentration of ammonium forrnate from 0.1M to 1.9M. The optical density of the efiluent was continuously read at 253 mpand the fractions containing a single substance were combined and lyophilized. Thus, the following components were obtained: CuAtl 33.9 mg. (300 pg/mg); CuAt2 220 mg. (644 pg/mg.); CuBt2 which contaminated in CuAt 7.2 mg. (2240 ug/mg.); CuAt3 15.1 mg. (671 Lg/mg); CuAt4 3.6 mg.; CuAtS 72.7 mg. (1460 pig/mg); CuBt4 (contamination in CuAt) 8.8 mg. (2800 ug/mgJ; CuAt6 3.5 mg. By the same procedure of CM Sephadex C-25 chromatography, starting from 1.908 g. of CuBt showing an activity of 345 Lg/mg, the following active components were obtained: Cu- Bt2 125.2 mg. 2240 rig/mg); CuBt4 50 mg. (2100 ug/mg) From other samples of CuBtl, CuBt3 or CuBtS was obtained in a small quantity together with CuBt2 and CuBt4.

EXAMPLE7 Bleomycin CuAt, 525.2 mg. showing an activity of 94 rig/mg. which was obtained by the process shown in Example 6, was dissolved in 40 ml. of 0. 1N HC! and extracted repeatedly with chloroform containing dithizone at 0.5 percent until no reddish violet color remained. After chloroform was removed, the solution was neutralized with Dowex-3 resin and lyophilized. Thus, copper was removed and a white powder (475 mg.) was obtained which showed an activity of 112 I s s- Bleomycin has a peak at 293-295 my. in its ultraviolet absorption spectrum. Acid hydrolysis in 6N HCl at b C. yielded more than eight ninhydrin positive products. One of them was crystallized and the following properties were observed: the analytical data suggested the formula of C H N O S or C H N O S max 293 mp. (E 490), yellow by ninhydrin reaction, pK 3.0 and 9.2. It is unstable at alkaline pH. On the basis of the ultraviolet absorption it must be a chromophoric amino acid contained in all bleomycins.

While in the foregoing specification various embodiments of this invention have been set forth in specific detail and elaborated for the purpose of illustration, it will be apparent to those skilled in the art that this invention is susceptible to other embodiments and that many of the details can be varied widely without departing from the basic concept and the spirit and scope of the invention.

We claim:

1. The process of producing a biologically' active complex, identified as bleomycin, which comprises cultivating Streptomyces verticillus ATTC 15003 at a temperature of about 25 C. to 35 C. for at least 2 days under submerged aerobic conditions in an aqueous assimilable carbohydrate solution containing an assimilable nitrogenous nutrient until the substantial amount of the said antibiotic is produced in said medium.

2. The process of producing a biologically active complex, identified as bleomycin A, which comprises cultivating Streptomyces verticillus ATIC 15933 at a temperature of about 25 C. to 35 C. for at least 2 days under submerged aerobic conditions in an aqueous as sirnilable carbohydrate solution containing an assimilable nitrogenous nutrient until the substantial amount of the said antibiotic is produced and then recovering from the broth the bleomycin A thus produced by adsorption on a cation exchange resin and subsequent elution therefrom and then separating the bleomycin A from the bleomycin B present therewith by chromatography, said bleomycin A being the fraction which exhibits an Rf value of 0.88-0.94 by paper chromatography using 10% NH Cl.

3. A biologically active complex identified as bleomycin A which is prepared by the process of claim 2 and which inhibits Gram-positive and Gram-negative bacteria, inhibits Ehrlich carcinoma, is soluble in water and methanol, and practically insoluble in ethanol, butanol, acetone, ethyl acetate, chloroform and benzene, forms salts with acids, chelates with copper, shows two ultraviolet absorption maxima at 254 my. and 295 my, gives positive Pauly, Ehrlich, Dragendorf tests, gives negative Sakaguchi, Fehling, Tollens and Molisch tests and shows Rf 088-094 by paper chromatography using 10% NH CI.

4. The process of producing a biologically active substance identified as bleomycin A2 which is one of the main active components of bleomycin A and which gives a negative ninhydrin reaction, gives positive Pauly, Ehrlich, Dragendorf and permanganate tests and negative Sakaguchi, Fehling, Tollens, ferric chloride and Molisch tests, contains C, H. N, S and O and exhibits the ultraviolet absorption spectrum shown in FIG. 3 and the infrared absorption spectrum shown in FIG. 4 after is has been purified as its copper-containing form designated Cu-At2 in said Figures, said process comprising cultivating Streptomyces verticilius ATIC 15003 at a temperature of about 25 C. to 35 C. for at least two days under submerged aerobic conditions in an aqueous assimilable carbohydrate solution containing an assimilable nitrogeneous nutrient until the substantial amount of the said antibiotic is produced and then recovering from the broth the bleomycin A2 thus produced by adsorption on a cation exchange resin and subsequent elution therefrom and then separating the bleomycin A2 from the other bleo mycins present therewith by chromatography as the fraction which exhibits in its copper-containing from an Rf of about 0.40 in thin layer chromotography using silica gel and percent ammonium acetate-methanol 1:1 and a negative Sakaguchi reaction.

5. A biologically active substance identified as bleomycin A2 which is prepared by the process of claim 4 and which gives a negative ninhydrin reaction, gives positive Pauly, Ehrlich, Dragendorf and permanganate tests and negative Sakaguchi, Fehling, Tollens, ferric chloride and Molisch tests, contains C, H, N, S and O and exhibits the ultraviolet absorption spectrum shown in FIG. 3 and the infrared absorption spectrum shown in FIG. 4 after it has been purified as its copper-containin g form designated Cu-AtZ in said Figures.

6. The process of producing a biologically active substance identified as bleomycin A5 in its copper-free form and as Cu-AtS in its copper-containing form which is one of the main components of bleomycin A and which gives a positive ninhydrin reaction, and gives positive Pauly, Ehrlich, Dragendorf and permanganate tests and negative Sakaguchi, Fehling, Tollens, ferric chloride and Mollisch tests, and exhibits the ultraviolet absorption spectrum shown in FIG. 3 and the infrared absorption spectrum shown in FIG. 4 after it has been purified as its copper-containing form designated Cu-At5 in said Figures, said process comprising cultivating Streptomyces verticillus ATCC 15003 at a temperature of about 25 C. to 35 C. for at least two days under submerged aerobic conditions in an aqueous assimilable carbohydrate solution containing an assimilable nitrogenous nutrient until the substantial amount of the said antibiotic is produced and then recovering from the broth the bleomycin A5 thus produced by adsorption on a cation exchange resin and subsequent elution therefrom and then separating the bleomycin A5 from the other bleomycins present therewith by chromatography as the fraction which exhibits in its copper-containing form an Rf of about 0.51 in thin layer chromotography using silica gel and 10 percent ammonium acetate-methanol 1 1 and a negative Sakaguchi reaction.

7. A biologically active substance identified as bleors sassarsz s ns is ch rs #5:: process of claim 6 and which gives a positive ninhydrin reaction, and gives positive Pauly, Ehrlich, Dragendorf and permanganate tests and negative Sakaguchi, Fehling, Tollens, ferric chloride and Molisch tests, and exhibits the ultraviolet absorption spectrum shown in FIG. 3 and the infrared absorption spectrum shown in FIG. 4 after it has been purified as its copper-containing form designated Cu-At5 in said Figures.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION 5, 91 Dated August 1, 1972 Hamao Umezawa, Kenji Maeda, Yoshiro Okami and Inventor(s) Tomio Takeuchi Patent No.

It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

In line 5 of Claim 2 for "A'ICC 15955 read "ATCC 15005.

In line 8 of Claim 3 for "25H" read "2 M" and in the last line for "NH Cl read "NI-I 61".

In line 2 of Claim 7 after "Cu-A135" delete 1".

Signed and sealed this 6th day of February 1973.

(SEAL) Attest:

EDWARD M.FLETCHE;R,JR. ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents 

2. The process of producing a biologically active complex, identified as bleomycin A, which comprises cultivating Streptomyces verticillus ATTC 15933 at a temperature of about 25* C. to 35* C. for at least 2 days under submerged aerobic conditions in an aqueous assimilable carbohydrate solution containing an assimilable nitrogenous nutrient until the substantial amount of the said antibiotic is produced and then recovering from the broth the bleomycin A thus produced by adsorption on a cation exchange resin and subsequent elution therefrom and then separating the bleomycin A from the bleOmycin B present therewith by chromatography, said bleomycin A being the fraction which exhibits an Rf value of 0.88- 0.94 by paper chromatography using 10% NH4Cl.
 3. A biologically active complex identified as bleomycin A which is prepared by the process of claim 2 and which inhibits Gram-positive and Gram-negative bacteria, inhibits Ehrlich carcinoma, is soluble in water and methanol, and practically insoluble in ethanol, butanol, acetone, ethyl acetate, chloroform and benzene, forms salts with acids, chelates with copper, shows two ultraviolet absorption maxima at 254 m Mu and 295 m Mu , gives positive Pauly, Ehrlich, Dragendorf tests, gives negative Sakaguchi, Fehling, Tollens and Molisch tests and shows Rf 0.88-0.94 by paper chromatography using 10% NH5Cl.
 4. The process of producing a biologically active substance identified as bleomycin A2 which is one of the main active components of bleomycin A and which gives a negative ninhydrin reaction, gives positive Pauly, Ehrlich, Dragendorf and permanganate tests and negative Sakaguchi, Fehling, Tollens, ferric chloride and Molisch tests, contains C, H. N, S and O and exhibits the ultraviolet absorption spectrum shown in FIG. 3 and the infrared absorption spectrum shown in FIG. 4 after is has been purified as its copper-containing form designated Cu- At2 in said Figures, said process comprising cultivating Streptomyces verticillus ATTC 15003 at a temperature of about 25* C. to 35* C. for at least two days under submerged aerobic conditions in an aqueous assimilable carbohydrate solution containing an assimilable nitrogeneous nutrient until the substantial amount of the said antibiotic is produced and then recovering from the broth the bleomycin A2 thus produced by adsorption on a cation exchange resin and subsequent elution therefrom and then separating the bleomycin A2 from the other bleomycins present therewith by chromatography as the fraction which exhibits in its copper-containing from an Rf of about 0.40 in thin layer chromotography using silica gel and 10 percent ammonium acetate-methanol (1:1) and a negative Sakaguchi reaction.
 5. A biologically active substance identified as bleomycin A2 which is prepared by the process of claim 4 and which gives a negative ninhydrin reaction, gives positive Pauly, Ehrlich, Dragendorf and permanganate tests and negative Sakaguchi, Fehling, Tollens, ferric chloride and Molisch tests, contains C, H, N, S and O and exhibits the ultraviolet absorption spectrum shown in FIG. 3 and the infrared absorption spectrum shown in FIG. 4 after it has been purified as its copper-containing form designated Cu-At2 in said Figures.
 6. The process of producing a biologically active substance identified as bleomycin A5 in its copper-free form and as Cu- At5 in its copper-containing form which is one of the main components of bleomycin A and which gives a positive ninhydrin reaction, and gives positive Pauly, Ehrlich, Dragendorf and permanganate tests and negative Sakaguchi, Fehling, Tollens, ferric chloride and Mollisch tests, and exhibits the ultraviolet absorption spectrum shown in FIG. 3 and the infrared absorption spectrum shown in FIG. 4 after it has been purified as its copper-containing form designated Cu- At5 in said FIgures, said process comprising cultivating Streptomyces verticillus ATCC 15003 at a temperature of about 25* C. to 35* C. for at least two days under submerged aerobic conditions in an aqueous assimilable carbohydrate solution containing an assimilable nitrogenous nutrient until the substantial amount of the said antibiotic is produced and then recovering from the broth the bleomycin A5 thus produced by adsorption on a cation exchange resin and subsequent elution therefrom And then separating the bleomycin A5 from the other bleomycins present therewith by chromatography as the fraction which exhibits in its copper-containing form an Rf of about 0.51 in thin layer chromotography using silica gel and 10 percent ammonium acetate-methanol (1.1) and a negative Sakaguchi reaction.
 7. A biologically active substance identified as bleomycin A5 in its copper-free form and as Cu- At5 4 l in its copper-containing form which is prepared by the process of claim 6 and which gives a positive ninhydrin reaction, and gives positive Pauly, Ehrlich, Dragendorf and permanganate tests and negative Sakaguchi, Fehling, Tollens, ferric chloride and Molisch tests, and exhibits the ultraviolet absorption spectrum shown in FIG. 3 and the infrared absorption spectrum shown in FIG. 4 after it has been purified as its copper-containing form designated Cu- At5 in said Figures. 